1. Field of the Invention
The invention is related to a method for amplifying a methylated target nucleic acid in a sample while avoiding amplification of a non-methylated target nucleic acid by inactivating it. This is accomplished by a restriction enzyme digest after bisulfite treatment of the target nucleic acid. The invention is further related to the use of a restriction enzyme to avoid amplification of a non-methylated target nucleic acid while amplifying a methylated target nucleic acid in a sample and kits for performing the methods according to the invention.
2. Description of the Related Art
Genes constitute only a small proportion of the total mammalian genome, and the precise control of their expression in the presence of an overwhelming background of noncoding deoxyribonucleic acid (DNA) presents a substantial problem for their regulation. Noncoding DNA, containing introns, repetitive elements, and potentially active transposable elements requires effective mechanisms for its long term silencing. Mammals appear to have taken advantage of the possibilities afforded by cytosine methylation to provide a heritable mechanism for altering DNA-protein interactions to assist in such silencing. DNA methylation is essential for the development of mammals; and plays a potential role during aging and cancer. The involvement of methylation in the regulation of gene expression and as an epigenetic modification marking imprinted genes is well established. In mammals, methylation occurs only at cytosine residues and more specifically only on cytosine residues adjacent to a guanosine residue, i.e. at the sequence CG. The detection and mapping of DNA methylation sites are essential steps towards understanding the molecular signals which indicate whether a given sequence is methylated.
This is currently accomplished by the so-called bisulfite method described by Frommer, M., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 1827-1831 for the detection of 5-methyl-cytosines. The bisulfite method of mapping 5-methylcytosine uses the effect that sodium hydrogen sulfite reacts with cytosine but not or only poorly with 5-methyl-cytosine. Cytosine reacts with bisulfite to form a sulfonated cytosine reaction intermediate being prone to deamination resulting in a sulfonated uracil which can be desulfonated to uracil under alkaline conditions. It is common knowledge that uracil has the base pairing behavior of thymine different to the educt cytosine whereas 5-methylcytosine has the base pairing behavior of cytosine. This makes the discrimination of methylated or non-methylated cytosines possible by e.g. bisulfite genomic sequencing (Grigg, G., and Clark, S., Bioessays 16 (1994) 431-436; Grigg, G. W., DNA Seq. 6 (1996) 189-198), methylation specific PCR (MSP) disclosed in U.S. Pat. No. 5,786,146 or by the use of blocking probes in PCR reactions (WO2002/072880). Oakeley, E. J., (Pharmacology & Therapeutics 84 (1999) 389-400. DNA methylation analysis: a review of current methodologies) reviews current methodologies of DNA methylation analysis.
There are various documents addressing specific aspects of the bisulfite reaction (Benyajati, C., et al., Nucleic Acids Res. 8 (1980) 5649-5667) make general investigations to the bisulfite modification of 5-methyl-deoxycytosine and deoxycytosine. Olek, A., et al., Nucleic Acids Res. 24 (1996) 5064-5066 disclose a method for bisulfite base sequencing whereby bisulfite treatment and subsequent PCR steps are performed on material embedded in agarose beads. In the bisulfite method as disclosed by Clark, S. J., et al., Nucleic Acids Res. 22 (1994) 2990-2997, the sample is desalted after deamination. Raizis, A. M., et al., Anal. Biochem. 226 (1995) 161-166 disclose a bisulfite method of 5-methylcytosine mapping that minimizes template degradation. They investigate the influence of pH, temperature and time of reaction. Similar investigations have been made by Grunau, C., et al., Nucleic Acids Res. 29 (2001) E65-5 or Warnecke, P. M., et al., Methods 27 (2002) 101-107. Different additional components in the bisulfite mixture are disclosed by WO 01/98528 or by Paulin, R. et al., Nucleic Acids Res. 26 (1998) 5009-5010. An additional bisulfite step after bisulfite treatment and PCR is disclosed in WO 02/31186. Komiyama, M., and Oshima, S., Tetrahedron Letters 35 (1994) 8185-8188 investigate the catalysis of bisulfite-induced deamination of cytosine in oligodeoxyribonucleotides. A specific bisulfite protocol is disclosed by WO 2004/067545. A variation of the bisulfite genomic sequencing method is disclosed by Feil, R., et al., Nucleic Acids Res. 22 (1994) 695-696, whereby the genomic DNA is bound to glass beads after deamination and washed. After elution the nucleic acid is desulfonated. EP 1 394 173 discloses a bisulfite method whereby the DNA is bound to the glass surface of a solid phase. Kits for performing bisulfite treatments are commercially available from Intergen, distributed by Serologicals Corporation, Norcross, Ga., USA, e.g. CpGenome™ DNA modification kit. The polymerase chain reaction (PCR) as described in U.S. Pat. No. 4,683,202 is also used in the field of the analysis of methylated nucleic acids. This method is even able to amplify analyte nucleic acids, e.g. of HCV, that are present in the smallest concentrations to such an extent that they become accessible to those nucleic acid tests which have been restricted to highly concentrated analytes. However, over time it has turned out that the laboratories in which the amplifications were carried out have in the meantime already become so strongly contaminated with the amplified nucleic acids that tests in samples which in fact do not contain the low concentrated nucleic acid at all lead to false-positive results since the samples have become contaminated by the environment with nucleic acids from previous amplifications (cross-contaminations). The high sensitivity of the amplification-based nucleic acid tests enables the detection of even the slightest contaminations and hence simulates the presence of the analyte in the sample (false-positive results).
EP-A-0 401 037 describes a method which partially remedies the described deficiency. In this method mononucleotides that are not naturally present in the nucleic acid to be detected are incorporated during the amplification into the amplificate of each analyte nucleic acid. Before a subsequent amplification is carried out, the sample together with the reagents used are subjected to a pretreatment in which all imported amplificates from earlier amplifications are enzymatically degraded. Uracil-N-glycosylase (UNG) is an example of a degradation reagent and dUTP is an example of a modified building block for the amplificates. An alternative method utilizes primers containing uracil instead of mononucleotides containing uracil. Such a method in which the primer binding sites are degraded on amplificates generated earlier is described in EP-A-0 415 755.
The mechanism of this decontamination method is based on the specific recognition of uracil-containing amplificates which are degraded by the enzyme. In the preparation of the amplification reaction UNG is added to the sample and usually already together with the master mix which contains all reagents necessary for the amplification. The aforementioned degradation reaction takes place in a brief incubation step before the subsequent amplification. If the reaction mixture is subsequently heated to a temperature above ca. 40° C., then UNG is inactivated. This is necessary to ensure that the UNG does not degrade the newly synthesized DNA which accumulates during the course of the amplification.
There are several documents disclosing further methods on the decontamination of mixtures used for this type of reaction. Klaschik, S. et al. (Molecular Biotechnology 22 (2002) 231-242. Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time PCR) disclose a comparison of decontamination methods using restriction enzyme cleavage compared with other methods. There is no methylation-specific cleavage and not in combination with bisulfite method. Only a DNAse digestion and not a restriction enzyme digestion was regarded to be effective. Abravaya, K. et al. (Lee, H. H. et al. (Ed.), Nucleic Acid Amplification Technologies 1997, 125-133. Strategies to avoid amplicon contamination) review techniques developed to prevent carryover contamination by contaminant DNA in DNA amplification procedures. The review includes pre-amplification decontamination using endonucleases but there is no disclosure of bisulfite modification or use thereof in methylation detection. U.S. Pat. No. 5,683,896 discloses another process for controlling contamination of nucleic acid and amplification reactions. US 2004/0005555 discloses the detection of bacteremia in emergency department patients at risk for infective endocarditis using universal 16S rRNA primers in a decontaminated PCR assay. The background DNA present in all PCR reagents is eliminated using a restriction endonuclease AluI digestion having multiple digestion sites in the amplicon but not in the primer sets. The restriction enzyme AluI enzyme is inactivated by heating to a temperature which inactivates AluI but not Taq polymerase. The method is not disclosed in combination with the bisulfite method and cannot be used when target DNA is present. DeFilippes, F. M. (Biotechniques 10 (1991) 26-30. Decontaminating the polymerase chain reaction) discloses the addition of template DNA to a modified PCR mixture to simulate contamination. The template DNA was inactivated by restriction enzyme digestion. After inactivation of restriction enzymes additional DNA template, buffer and Taq polymerase were added to the reaction and PCR proceeded. Limitations of this method are discussed. There is no disclosure of the bisulfite treatment or restriction enzyme digestion.
Several documents describe the general combination of bisulfite technology and restriction enzyme cleavage in the field of detection of methylated nucleic acids. Sadri, R. et al. (Nucleic Acids Res. 24 (1996) 5058-5059, Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification) disclose the bisulfite treatment of DNA and subsequent amplification. Changes in restriction enzyme sites are detected. Velinov, M. et al. (Methods in Molecular Biology 217 (2003) 209-216, PCR-based strategies for the diagnosis of Pradet-Willi/Angelman syndromes) disclose a PCR-based methylation test using methylation-specific digestion of the amplified, bisulfite-treated DNA. Velinov, M. et al. (Molecular Genetics and Metabolism 69 (2000) 81-83. The feasibility of PCR-based diagnosis of Prader-Willi and Angelman syndromes using restriction analysis after bisulfite modification of genomic DNA) disclose a PCR-based methylation test using methylation-specific digestion of the amplified, bisulfite-treated DNA. Xiong, Z. et al. (Nucleic Acids Res. 25 (1997) 2532-2534. COBRA: a sensitive and quantitative DNA methylation assay) uses restriction enzyme digestion to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite treated DNA. WO2003/000926 discloses the bisulfite treatment of DNA and subsequent amplification. Thereafter, the amplificate is digested with restriction endonucleases. The enzyme resistant fraction of digested DNA is then amplified in a further PCR. In all these documents, restriction enzyme cleavage is performed after PCR amplification.
Several other documents describe restriction enzyme digestions in connection with the bisulfite method. Fojtova, M. et al. (Plant Science 160 (2001) 585-593. Cytosine methylation of plastid genome in higher plants. Fact or artifact?) disclose bisulfite genomic sequencing performed on EcoRII-restricted DNA difference. The restriction enzyme digestion is performed before bisulfite treatment. Clark, S. J. et al. (Ed. G. R: Taylor, Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA (1997) 151-162, Publisher CRC, Boca Raton, Fla.) review current methods available for the study of cytosine methylation in genomic DNA. The restriction enzyme digestions and the bisulfite method are described independently similarly to WO2003/064701. Jang, K.-H. et al. (J. Microbiology and Biotechnology 11 (2001) 819-824. Identification of a sequence containing methylated cytidine in Corynebacterium glutamicum and Brevibacterium flavum using bisulfite DNA derivatization and sequencing) disclose the bisulfite treatment of DNA and subsequent amplification, restriction enzyme digestion to remove not fully converted DNA and another round of amplification. Then, the DNA sequence is determined.
Other documents describe the use of restriction enzyme digests for the analysis of methylations. Kaneda, S. A. et al. (Molecular Medicine 39 (2002) 824-832) review various methods for analysis of DNA methylation including restriction enzyme digestions. WO2003/027259 discloses assays for detecting DNA methylation associated with diseases in mouse and their use in diagnosis. The technique to detect the extent of DNA methylation entails generating DNA fragments of a test sample by cleaving at methylation sites that are not methylated while sparing methylation sites in the DNA that are methylated. The method is not used in combination with the bisulfite method. Moore, T. (Methods in Molecular Biology 181 (2002) 193-203. Southern analysis using methyl-sensitive restriction enzymes) discloses the use of methyl-sensitive restriction enzymes and Southern analysis but not in combination with the bisulfite method. EP 0 976 835 discloses the detection of nucleic acid methylation using amplification fragment length polymorphism which is not used in combination with the bisulfite method. Pogribny, I. et al. (Biochem. Biophys. Res. Commun. 262 (1999) 624-628. Sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands) disclose a method based on the use of methylation-sensitive restriction endonucleases that leave a 5′-guanine overhang after DNA cleavage, with subsequent single nucleotide extension with radiolabeled [3H]dCTP. The method is not combined with the bisulfite method or PCR amplification. Kupper, D. et al. (Biotechniques 23 (1997) 843-847. Reliable detection of DNA CpG methylation profiles by the isoschizomers MspI/HpaII using oligonucleotide stimulators) disclose a protocol for detecting CpG methylation by the isoschizomeric restriction endonucleases MspI/HpaII but not in combination with the bisulfite method. Watts, G. S. et al. (Nucleic Acids Res. 23 (1995) 4740-4741. Detecting differences in 5-methylcytosine using restriction enzyme isoschizomers: an endogenous control for complete digestion) disclose the southern blot analysis of genomic DNA cut with methylation-sensitive isoschizomers like MspI/HpaII but not in combination with the bisulfite method. Chang, S. et al. (Plant Molecular Biology Reporter 10 (1992) 362-366. PCR amplification following restriction to detect site-specific methylation) disclose a procedure to test for DNA methylation at sites recognized by methylation-sensitive restriction endonucleases. The procedure is based on the assumption that PCR will amplify sequences between two primers only if target DNA is intact after digestion. The method is not used in combination with the bisulfite method. Szyf, M. et al. (Nucleic Acids Res. 10 (1982) 7247-7259. Studies on the biological role of DNA methylation: V—The pattern of E. coli DNA methylation) disclose an analysis of the state of the methylation of GATC sites in newly replicating DNA using the restriction enzyme DpnI but not in combination with the bisulfite method. Youssoufian, H. et al. (J. Mol. Biol. 150 (1981) 133-136). Detection of methylated sequences in eukaryotic DNA with the restriction endonucleases SmaI and XmaI) disclose two isoschizomers which either digest specific CpG sites methyl-sensitively or not. The restriction enzymes are not used in combination with the bisulfite method. Cedar, H. et al. (Nucleic Acids Res. 6 (1979) 2125-2132. Direct detection of methylated cytosine in DNA by use of restriction enzyme MspI) disclose the analysis of the state of methylation of CCGG sites using MspI/HpaII restriction enzymes and subsequent gel analysis but not in combination with the bisulfite method.